About TSRI
Research & Faculty
News & Publications
Scientific Calendars
Scripps Florida
PhD Program
Campus Services
Work at TSRI
TSRI in the Community
Giving to TSRI
Directory
Library
Contact
Site Map & Search
TSRI Home

The Skaggs Institute
for Chemical Biology


Scientific Report 2007




Insights Into Protein Chemistry and Biology From Protein Structure


E.D. Getzoff, A.S. Arvai, D.P. Barondeau, E.D. Garcin, C. Hitomi, K. Hitomi, M.D. Kroeger, A.J. Pratt, D.S. Shin, J.L. Tubbs, T.I. Wood

We investigate the interface between protein structural chemistry and biology. We focus on proteins involved in reactive oxygen defenses and in light interactions for photoactivation and signaling. We determine high-resolution crystallographic structures and combine these with solution analyses via hydrogen-deuterium exchange mass spectrometry and x-ray scattering to probe conformational and dynamic changes. On the basis of our integrated structural results, we propose comprehensive mechanistic models that explain how proteins function as efficient catalysts and molecular machines. We test these hypotheses with biochemical and mutational analyses to improve understanding of how proteins achieve and regulate their activities and to aid in applications of this knowledge for the design of proteins and inhibitors.

This year, in our Skaggs research, we achieved advances and published articles on initiation of neuronal disease by copper, zinc superoxide dismutase (SOD) mutations, regulation of nitric oxide synthase (NOS) and the role of this enzyme in neuronal cell death, structural chemistry controlling posttranslational modifications in green fluorescent protein, and roles for active-site histidines in (6-4) photolyase, which uses light to repair DNA damage.

Chemical Biology and Regulation of SOD And NOS

Skaggs funding significantly aids our investigations of the reactive oxygen control enzymes copper, zinc SOD and NOS. Specifically, we analyze the structural chemistry of these enzymes to help bridge the gap from protein structures to enzyme activities in vivo. For SOD, we designed a zinc-free variant of the human enzyme to help test the role of zinc binding and loss in the initiation of the fatal neurodegenerative disease familial amyotrophic lateral sclerosis, which is linked to more than 130 different mutations in human SOD. Our results, obtained in collaboration with J.A. Tainer, the Skaggs Institute, support the importance of the stable SOD core structure in preventing amyloid formation and toxic effects.

For NOS, we recently reported the biphasic coupling of neuronal NOS phosphorylation to N-methyl-D-aspartate receptors to regulate trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, synaptic plasticity, and neuronal cell death, in collaboration with G. Rameau, Johns Hopkins School of Medicine, Baltimore, Maryland, and E.B. Ziff, New York University, New York, New York.

The 3 human NOS isozymes offer key therapeutic targets for neurotransmission (neuronal NOS), regulation of blood pressure (endothelial NOS), and the immune response (inducible NOS). These highly similar, but differently regulated, isozymes all synthesize the diatomic molecule nitric oxide, which is both a molecular signal (at low concentrations) and a cytotoxin (at high concentrations). Our crystallographic structures delineate the NOS oxygenase module, reductase module, and calmodulin-bound linker; characterize their cofactor binding; and identify mechanisms for their functions in the synthesis and regulation of nitric oxide. In collaboration with Dr. Tainer, we are using small-angle x-ray scattering to test our assembly and mechanistic models for NOS by defining the shapes and conformational changes of NOS domains and their assemblies.

The aims of these ongoing cross-disciplinary mutational, biochemical, and structural investigations of NOS are to (1) determine the bases for functional domain interactions, cofactor recognition, and tuning for electron transfer and catalysis; (2) characterize the diverse regulatory mechanisms that differentially control the NOS isozymes; and (3) elucidate distinguishing features for isozyme-specific inhibitors. Isozyme-specific NOS inhibitors are sought for medicinal purposes and for advancing understanding of basic human physiology, but present a huge challenge because of active-site conservation. We are now making strong progress in defining the basis for isozyme-specific NOS inhibitors (Fig. 1).

Fig. 1. Isozyme-specific inhibition of NOS. Aminopyridine inhibitor binding to human inducible NOS (top) induces a cascade of side-chain conformational changes leading to the opening of a new selectivity pocket. In contrast, similar binding of the same aminopyridine inhibitor to endothelial NOS (bottom) is prevented by bulky amino acids (circled) distant from the active-site substrate-binding pocket above the heme.

Signal Transduction in Photoactive Proteins

We use Skaggs support to discover how living things use cofactor-protein partnerships to transduce environmental changes into appropriate biological responses. We are examining and testing the mechanisms of light-induced protein activities in the family of green and red fluorescent proteins used as biological markers; in the blue-light receptor photoactive yellow protein (PYP); and in the cryptochrome flavoproteins, which are components of circadian clocks in animals and humans, and their structural analogs, the photolyases that repair damaged DNA bases.

For green and red fluorescent proteins, our high-resolution crystallographic structures and mutagenesis results reveal the detailed structural chemistry for the posttranslational modifications these proteins can create at their active sites. This chemistry is surprisingly powerful. In fact, we tested our understanding by modifying the active site of green fluorescent protein to achieve radical cleavage of a carbon-carbon bond. We are now completing analyses of differences between green and red fluorescent proteins in their active sites and protein assemblies.

PYP is the prototype for the ubiquitous Per-Arnt-Sim (PAS) domains of proteins that mediate intraprotein and interprotein interactions and conformational changes in response to light, oxygen, redox potential, and small-molecule ligands. Our research indicates how light activation of PYP breaks the dark-state hydrogen-bonding network to trigger an allosteric T(tense)-to-R(relaxed) state conformational transition for signaling. We plan to test our proposed signal transduction mechanism within the structurally conserved PYP/PAS fold.

We identified and characterized the CryDASH cryptochrome protein family, which occurs in animal, plant, and bacterial species (in contrast with classic animal- and plant-specific cryptochromes). Cryptochromes share with the homologous light-activated DNA repair photolyases not only the overall protein fold but also the redox-active FAD cofactor bound in an unusual U-shaped conformation and the surrounding positive electrostatic surface consistent with a function in DNA binding. Through structural and functional studies of diverse members of the cryptochrome/photolyase families, we are deciphering how their similarities and differences direct the same cofactor and protein fold to produce different biological responses to light.

Publications

Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. The case of the missing ring: radical cleavage of a carbon-carbon bond and implications for GFP chromophore biosynthesis. J. Am. Chem. Soc. 129:3118, 2007.

Hill, N.J., Stotland, A., Solomon, M., Secrest, P., Getzoff, E., Sarvetnick, N. Resistance of the target islet tissue to autoimmune destruction contributes to genetic susceptibility in type 1 diabetes. Biol. Direct 2:5, 2007.

Rameau, G.A., Tukey, D.S., Garcin-Hosfield, E.D., Titcombe, R.F., Misra, C., Khatri, L., Getzoff, E.D., Ziff, E.B. Biphasic coupling of neuronal nitric oxide synthase phosphorylation to the NMDA receptor regulates AMPA receptor trafficking and neuronal cell death. J. Neurosci. 27:3445, 2007.

Roberts, B.R., Tainer, J.A., Getzoff, E.D., Malencik, D.A., Anderson, S.R., Bomben, V.C., Meyers, K.R., Karplus, P.A., Beckman, J.S. Structural characterization of zinc-deficient human superoxide dismutase and implications for ALS. J. Mol. Biol. 373:877, 2007.

Schleicher, E., Hitomi, K., Kay, C.W., Getzoff, E.D., Todo, T., Weber, S. Electron nuclear double resonance differentiates complementary roles for active site histidines in (6-4) photolyase. J. Biol. Chem. 282:4738, 2007.

Yamamoto, J., Tanaka, Y., Hitomi, K., Getzoff, E.D., Iwai, S. Spectroscopic studies on a novel intramolecular hydrogen bond within the (6-4) photoproduct. Nucleic Acids Symp. Ser. (Oxf.) 79, 2007. Issue 51.

 

Elizabeth D. Getzoff, Ph.D.
Professor

Getzoff Web Site